The invention relates to a drug target system for the identification of novel and highly specific antimicrobials. In particular, the present invention relates to screening assays to identify antimicrobial agents which uncouple or inhibit the bacterial phosphotransferase system.
Infectious diseases affect all age groups and cause one- third of all death worldwide. Respiratory infections, diarrheal diseases and tuberculosis, neonatal tetanus and whooping cough are the most prominent among the bacterial infections. Infectious diseases are still rising due to population growth, urbanization, and not least due to the widespread use of antibiotics that led to the development of antibiotic resistance. The increasing incidence of bacterial drug-resistance requires that new targets of antimicrobial therapy be identified and that inhibitors of these targets be discovered.
The present invention relates to a drug target system for novel and specific antimicrobials which modulate the bacterial phosphotransferase system. In particular, the present invention relates to the identification of agents which uncouple or inhibit the bacterial phosphotransferase system. In a preferred embodiment of the present invention, the assays to identify antimicrobial agents which
a) use a phosphoryl group acceptor which is not part of the phosphotransferase system and preferably hydrolyse spontaneously or under the influence of non-specific endogenous phosphohydrolases thereby uncoupling the phosphotransferase system, or
b) inhibit the phosphotransferase system activity by blocking or inactivating at least one of the enzymes of the phosphotransferase system.
The present invention also relates to screening assays to identify potential antimicrobial agents which screen for those agents which utilize a phosphoryl group that is not part of the bacterial phosphotransferase system, or agents which hydrolyse, spontaneously or under the influence of non-specific endogenous phosphohydrolases, thereby uncoupling the system. The present invention further relates to screening assays to identify agents which inhibit at least one of the enzymes of the phosphotransferase system.
The present invention further relates to a screening assay for the identification of chemical compound (peptide or specific small organic compound) which acts as an antimicrobial by inhibiting or uncoupling enzyme I comprising:
a) adding a test compound to a reaction mixture containing enzyme I and phosphoenolpyruvate; and
b) measuring pyruvate levels in the presence of lactate dehydrogenase and NADH, where increased levels of pyruvate serve as an indication that the test compound has uncoupling or inhibitory activity of enzyme I of the bacterial phosphotransferase system.
In a preferred embodiment, the molecular weight of the specific small organic compound is under 1,500.
Furthermore, the invention relates to novel antimicrobials identified by using said drug target system.